Currently, pathologists are performing microscopic examinations of various forms of tissue and cells. The microscopic image reveals information on morphology (e.g. cell types, differentiation, etc.), which is the basis for the diagnosis. In case the pathologist wants to examine the tissue/cells in more detail, biomarker-specific staining protocols are used, such as ER, PR, and HER2 in the case of breast cancer. Certain molecular tests are carried out in situ by so-called in-situ hybridization on DNA and RNA. The number of targets however is limited in such an approach. For improved stratification, molecular diagnostic tests are carried out to test gene expression (e.g. OncoTypeDX, Mammaprint) or to look for actionable mutations, like KRAS, EGFR, etc. with the aid of sequencing (e.g. Sanger or next generation sequencing) and expression profiling (e.g. micro-arrays, RNAseq, RT-PCR).
Tumor tissue is heterogeneous in composition and surrounded by stromal and infiltrating immune cells. This spatial heterogeneity can affect the molecular analysis, as the DNA and RNA of the tumor cells is diluted by DNA and RNA from cells that are not targeted by the analysis. To overcome this problem regions of interest (ROI) of the tissue/cells are selected on the slide and removed from the slide, for example, by scraping or (laser capture) micro dissection techniques, to be processed and analyzed in molecular assays and/or proteomic assays (protein compositions etc.). While collecting ROIs only limited positional information is stored and often the collected material is originating from different ROIs or ROIs are pooled and subsequently analyzed together. In case of laser capture micro dissection, small regions can be collected separately, but this is time consuming and does not allow for systematic analysis of the tissue/cells. Molecular analyses are time consuming and expensive. For a higher resolution mapping of molecular profiles this results in a large number of molecular tests with costs that would be too high for clinical practice. Tumor heterogeneity and tissue architecture may furthermore be a potential diagnostic parameter as they can provide clues about the clonal evolution of the tumor and its aggressiveness. Thus by removing ROIs from the slide in order to perform further molecular analyses this information is lost.
Heterogeneity maps have been created based on in-situ staining for molecular and protein biomarkers. See for example Almendro, et al., 2014, Cell Reports, 6: 514-527. A different approach was described by Armani et al., Lab Chip, 2009, 9(24): 3526-3534 and Armani et al., Anal Bioanal Chem, 2011, 400: 3383-3393, where a tissue slice was pressed into a well plate where in each well a single qPCR or RT-qPCR reaction was performed. Subsequently, a 2D map was generated of the amplified target.
US20140066318 A1 describes a probe array on a substrate onto which a tissue sample is placed. The probes bind to target nucleic acids and bound probe and target nucleic acid are extracted and used for molecular diagnosis.
In the present application we describe an approach that allows a high spatial resolution mapping of nucleic acids without sacrificing the degree of multiplexing that is available from next-generation sequencing by efficiently using multiplexing ROIs per sample. The method of the invention is based on the application of patterns of oligonucleotide probes comprising a barcode sequence taking into account tissue information, wherein the oligonucleotide probes bind to the nucleic acids in the sample. Various application technologies can be used and different ways of patterning can be employed, like a regular array with a certain pitch or alternatively an object-based patterning with defined regions of interest without shape constraints.
A key aspect of the invention is the identification of a ROI and defining the spatial resolution of the pattern individually based on the tissue information and question to be answered. The region of interest as well as the spatial resolution of the pattern can be chosen in response to image-based analysis of the tissue before the application of the reagents. By identification of at least one ROI and only providing oligonucleotide probes to the ROI, fewer species of oligonucleotide probes are required to provide a spatial map. Further, designing spatial resolution patterns individually without being restricted to an array format allows the number of oligonucleotide probes to be used to be determined on an individual basis. The method of the invention is therefore much cheaper than common prior art methods and compatible with selective profiling.
Another advantage of the method of the invention is that it fits smoothly in the current digital pathology workflow. The common digital pathology workflow is:
1. preparation of a hematoxylin and eosin stain staining slide,
2. scanning the hematoxylin and eosin stain slide to obtain a digital image
3. perform an image analysis to arrive at a pathological diagnosis (benign or malignant), and
4. concurrently identify ROIs for further molecular diagnosis to provide more precise diagnosis results.